A new approach to single molecule DNA sequencing is proposed. Single molecule methods can be applied to DNA isolated directly from a subject organism, eliminating the need to clone, map and sort DNA fragments prior to sequencing. The need for electrophoretic separation is eliminated and reagent consumption is minimal, promising unprecedented low cost. Single molecule detection (SMD) has been developed and practiced by physicists and chemists over the past 10 years. The crucial optical and detection systems needed for single molecule sequencing now exit. Research into single molecule sequencing has been limited to an exonuclease approach, where DNA labeled at every base position is digested and the released nucleotides are detected individually. Difficulties with this approach have centered on the inability of polymerases and exonuclearses to function efficiently on DNA labeled with bulky fluorophores at every base. We propose methods for circumventing these problems. The success of this project will create a new paradigm in DNA sequencing technology. PROPOSED COMMERCIAL APPLICATION: A genomic DNA sequencing technology that eliminates the need for cloning, physical mapping and electrophoresis would have tremendous appeal to the academic and industrial genomics research community.